Supercharge Your Innovation With Domain-Expert AI Agents!

Method for reducing ammonium and lactate production in cho cells

Active Publication Date: 2019-07-04
DANMARKS TEKNISKE UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Embedded in this patent is a method to create mammalian cells that have a faster growth rate, produce more biomass, and can express foreign proteins faster. The method involves knocking out genes involved in the breakdown of amino acids, which reduces the production of toxic byproducts and allows the cells to grow more quickly. This results in a higher yield of biomass and faster expression of foreign proteins.

Problems solved by technology

A central problem in CHO cell bioprocessing is accumulation of toxic metabolic by-products, which inhibit growth and impair product quality (Lao & Toth, 1997; Brodsky et al., 2014), leading to an overall reduced yield of high quality protein.
Finally, reactive aldehydes produced in the catabolic pathway of L-lysine, the saccharopine pathway, are potentially toxic and can form adducts and condensation products with small molecules (e.g. amines), proteins and DNA (Hallen et al., 2013).

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for reducing ammonium and lactate production in cho cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0051]Metabolic Network Reconstruction

[0052]A draft network reconstruction of the glycolytic and amino acid catabolic pathways in CHO cells was generated using the mouse metabolic pathways as template. Biochemical pathway data from mouse metabolism was retrieved from the Kyoto Encyclopedia of Genes and Genomes database (Kanehisa and Goto, 2000; Kanehisa et al., 2014) and homologous gene sequences in the CHO genome were identified using the Chinese hamster genome database www.CHOgenome.org (Hammond et al., 2012). The draft network reconstruction was further refined by careful curation of gene-protein-reaction relationships using manual genome annotation and literature evidence. The finalized reconstruction featured 319 proteins catalyzing 183 reactions with 188 metabolites.

[0053]Single-Guide RNA Target Design and Transfection

[0054]Design and selection of single-guide RNA (sgRNA) target sites was performed with the online tool “CRISPy” (Ronda et al., 2014). The sgRNA expression vector...

example 2

Knock Out OF HPD in a IgG-Producing Cell Line

[0064]Single-Guide RNA Target Design and Transfection

[0065]Design and selection of single-guide RNA (sgRNA) target sites was performed with the online tool “CRISPy” (Ronda et al., 2014). The sgRNA expression vectors were constructed as previously described (Ronda et al., 2014). Prior to transfection, CHO-S suspension cells obtained from Life Technologies, transformed to express an IgG and selected for high specific productivities, were grown in CD-CHO medium supplemented with 8 mM L-glutamine (Gibco) and 0.5% anti-clumping reagent (Gibco) in a humidified shaking incubator operated at 37° C., 5% CO2 and 120 rpm. One day prior to transfection, cells were washed and seeded in medium without anti-clumping reagent at 5×105 cells / mL. Cells were transfected with expression vectors encoding GFP_2A-Cas9 (Gray et al., 2015) and sgRNA targeting Gad2 to generate single-gene knock out transfectants. For controls, “mock” cell lines were generated trans...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Fractionaaaaaaaaaa
Fractionaaaaaaaaaa
Fractionaaaaaaaaaa
Login to View More

Abstract

The present invention relates to modified producer cells for improved production of therapeutic proteins. Specifically, the inventors have found that removing genes involved in amino acid catabolism in Chinese Hamster Ovary (CHO) cells improves the cell growth and viability and likely also the yield of a recombinant therapeutic protein produced by the cells.

Description

FIELD OF THE INVENTION[0001]The present invention relates to modified producer cells for improved production of therapeutic proteins. Specifically, the inventors have found that removing genes involved in amino acid catabolism in Chinese Hamster Ovary (CHO) cells improves the cell growth and viability and likely also the yield of a recombinant therapeutic protein produced by the cells.BACKGROUND OF THE INVENTION[0002]CHO cells are the predominant cell factory for production of recombinant therapeutic proteins, a segment of the pharmaceutical industry worth more than 140 billion USD in 2013 alone (Walsh, 2014). A central problem in CHO cell bioprocessing is accumulation of toxic metabolic by-products, which inhibit growth and impair product quality (Lao & Toth, 1997; Brodsky et al., 2014), leading to an overall reduced yield of high quality protein. CHO cells mainly acquire energy for growth and protein synthesis from glucose and glutamine catabolism, however evidence suggest that ot...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12P21/02C12N15/52C12N5/071
CPCC12P21/02C12N15/52C12N5/0682C12N9/0069C12Y113/11027
Inventor LEY, DANIELANDERSEN, MIKAEL RORDAMKILDEGAARD, HELENE FAUSTRUP
Owner DANMARKS TEKNISKE UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com